Diabetes is a major public health problem. By 1998, 16 million Americans had been diagnosed as having diabetes (American Diabetes Association, 1998).
Ocular complications of diabetes are the leading cause of new cases of legal blindness in people ages 20 to 74 in the United States. The risk for lower extremity amputation is 15 times greater in individuals with diabetes than in individuals without it. Kidney disease is a frequent and serious complication of diabetes. Approximately 30 percent of all new patients in the United States being treated for end-stage renal disease have diabetes. Individuals with diabetes are also at increased risk for periodontal disease. Periodontal infections advance rapidly and lead not only to loss of teeth but also to compromised metabolic function. Women with diabetes risk serious complications of pregnancy. Current statistics suggest that the mortality rates for infants of mothers with diabetes is approximately 7 percent.
Clearly, the economic burden of diabetes is enormous. Each year, patients with diabetes or its complications spend 24 million patient-days in hospitals. Diabetes is our nation""s most expensive disease with an estimated total annual cost of $98 billion; however, the full economic impact of this disease is even greater because additional medical expenses often are attributed to the specific complications of diabetes rather than to diabetes itself.
Diabetes is a chronic, complex metabolic disease that results in the inability of the body to properly maintain and use carbohydrates, fats, and proteins. It results from the interaction of various hereditary and environmental factors and is characterized by high blood glucose levels caused by a deficiency in insulin production or an impairment of its utilization. Most cases of diabetes fall into two clinical types: Type I, or juvenile-onset, and Type II, or adult-onset. Type I diabetes is often referred to as Insulin Dependent Diabetes, or IDD. Each type has a different prognosis, treatment, and cause.
Approximately 5 to 10 percent of diabetes patients have IDD. IDD is characterized by a partial or complete inability to produce insulin usually due to destruction of the insulin-producing xcex2 cells of the pancreatic islets of Langerhans. Patients with IDD would die without daily insulin injections to control their disease.
Few advancements in resolving the pathogenesis of diabetes were made until the mid-1970s when evidence began to accumulate to suggest that Type I IDD had an autoimmune etiopathogenesis. It is now generally accepted that IDD results from a progressive autoimmune response which selectively destroys the insulin-producing xcex2 cells of the pancreatic Islets of Langerhans in individuals who are genetically predisposed. Autoimmunity to the xcex2 cell in IDD involves both humoral (Baekkeskov et al., 1982; Baekkeskov et al., 1990; Reddy et al. 1988; Pontesilli et al., 1987) and cell-mediated (Reddy et al. 1988; Pontesilli et al., 1987; Wang et al., 1987) immune mechanisms. Humoral immunity is characterized by the appearance of autoantibodies to xcex2 cell membranes (anti-69 kD and islet-cell surface autoantibodies), xcex2 cell contents (anti-carboxypeptidase A1, anti-64 kD and/or anti-GAD autoantibody), and/or xcex2 cell secretory products (anti-insulin). While serum does not transfer IDD, anti-xcex2 cell autoantibody occurs at a very early age, raising the question of an environmental trigger, possibly involving antigenic mimicry. The presence of cell-mediated immunological reactivity in the natural course of IDD is evidenced by an inflammatory lesion within the pancreatic islets, termed insulitis. Insulitis, in which inflammatory/immune cell infiltrates are clearly visible by histology, has been shown to be comprised of numerous cell types, including T and B lymphocytes, monocytes and natural killer cells (Signore et al., 1989; Jarpe et al., 1991). Adoptive transfer experiments using the NOD (non-obese diabetic) mouse as a model of human IDD have firmly established a primary role for auto-aggressive T lymphocytes in the pathogenesis of IDD (Bendelac, et al., 1987; Miller et al., 1988; Hanafusa et al., 1988; Bendelac et al., 1988). Unfortunately, the mechanisms underlying destruction of the pancreatic xcex2 cells remain unknown.
Recent efforts to culture pancreatic cells, including efforts reported in the following publications, have focused on cultures of differentiated or partially differentiated cells which in culture have grown in monolayers or as aggregates. By contrast to these reports, the instant invention discloses a method and a structure wherein an islet-like structure is produced which has a morphology and a degree of cellular organization much more akin to a normal islet produced in vivo through neogenesis.
Gazdar, et al. (1980), disclosed a continuous, clonal, insulin- and somatostatin-secreting cell line established from a transplantable rat islet cell tumor. However, the cells disclosed were tumorigenic and were not pluripotent.
Brothers, A. J. (WO 93/00441, 1993), disclosed hormone-secreting cells, including pancreatic cells, maintained in long-term culture. However, the cells cultured are differentiated, as opposed to pluripotent stem cells, which are selected at an early stage for their hormone secreting phenotype, as opposed to their capacity to regenerate a pancreas-like structure.
Korsgren, et al. (1993), disclosed an in vitro screen of compounds for their potential to induce differentiation of fetal porcine pancreatic cells. The instant invention does not depend on the use of fetal tissue.
Nielsen, J. H. (WO 86/01530, 1986), disclosed a method for proliferation of wholly or partially differentiated beta cells. However, this disclosure depended on fetal tissue as a source of the islet cells grown in culture.
McEvoy et al. (1982), disclosed a method for tissue culture of fetal rat islets and compared the effect of serum on the defined medium maintenance, growth and differentiation of A, B, and D cells. Once again, the source of islet cells is fetal tissue.
Zayas et al. (EP 0 363 125, 1990), disclosed a process for proliferation of pancreatic endocrine cells. The process depends on the use of fetal pancreatic tissue, and a synthetic structure, including collagen which is prepared to embed these cells for implantation. The thus produced aggregates of cultured cells upon implantation require 60-90 days before having any effect on blood glucose levels, and require 110-120 days before euglycemia is approached. In contrast, the instant invention provides in vitro grown islet-like structures which do not require collagen or other synthetic means for retention of their organization, and which, upon implantation, provide much more rapid effects on the glycemic state of the recipient.
Coon et al. (WO 94/23572, 1994), disclosed a method for producing an expanded, non-transformed cell culture of pancreatic cells. Aggregated cultured cells are then embedded in a collagen matrix for implantation, with the attendant shortcomings noted for the Zayas et al., EP 0 363 125, structures and the distinctions noted with the structure produced according to the instant invention.
In view of the foregoing reports, the instant invention, wherein functional islet-like structures containing cells which express insulin, glucagon and/or somatostatin which can be implanted into clinically diabetic mammals which subsequently remain healthy (after elimination of insulin treatment), is surprising. This is because conventional and immunofluorescent histology of the pancreatic islets of Langerhans (Lacey et al, 1957; Baum et al., 1962; Dubois, 1975; Pelletier et al., 1975; Larsson et al., 1975), together with recent three dimensional imaging (Brelje et al., 1989), have revealed a remarkable architecture and cellular organization of pancreatic islets that is ideal for rapid, yet finely controlled, responses to changes in blood glucose levels. It could not be predicted that such a structure could be produced in vitro, particularly when one considers that during embryogenesis, islet development within the pancreas appears to be initiated from undifferentiated precursor cells associated primarily with the pancreatic ductal epithelium (Pictet et al., 1972) i.e. non-islet cells. The ductal epithelium rapidly proliferates, then subsequently differentiates into the various islet-associated cell populations (Hellerstrom, 1984; Weir et al., 1990; Teitelman et al., 1993; Beattie et al., 1994). The resulting islets are organized into spheroid structures in which insulin-producing xcex2 cells form a core surrounded by a mantle of non-xcex2 cells. For the most part, glucagon-producing a cells (if the islet is derived from the dorsal lobe) or alternatively, pancreatic peptide-producing, PP cells (if the islet is derived from the ventral lobe), reside within the outer cortex (Brelje et al., 1989; Weir et al., 1990). Somatostatin-producing xcex4 cells, which are dendritic in nature, reside within the inner cortex and extend pseudopodia to innervate the xcex1 (or PP) cells and the xcex2 cells. These spheroid islet structures tend to bud from the ductal epithelium and move short distances into the surrounding exocrine tissue. Angiogenesis-induced vascularization results in direct arteriolar blood flow to mature islets (Bonner-Weir et al., 1982; Teitelman et al., 1988; Menger et al., 1994). Since blood glucose can stimulate xcex2 cell proliferation, vascularization may act to increase further the numbers of xcex2 cells. Similarly, neurogenesis leads to the innervation of the islets with sympathetic, parasympathetic and peptidergic neurons (Weir et al. , 1990). That we have been able to produce functional islet-like structures in vitro which can then be implanted to produce pancreas-like structures, is therefore quite remarkable.
Unfortunately, the cellular organization of the islet can be destroyed in diseases such as type I, insulin dependent diabetes (IDD), in which a progressive humoral and cell-mediated autoimmune response results in specific destruction of the insulin-producing xcex2 cells (Eisenbarth, 1986; Leiter et al., 1987). Because the xcex2 cell is considered to be, for the most part, a differentiated end-stage cell, it is believed that the body has limited capacity to generate new xcex2 cells, thus necessitating regular life-long insulin therapy once the xcex2 cell mass is destroyed. However, in experimental animals, the xcex2-cell mass has been shown to increase and decrease in order to maintain euglycemia (Bonner-Weir et al., 1994). This plasticity can occur through two pathways of islet growth: first, by neogenesis, or growth of new islets by differentiation of pancreatic ductal epithelium, and second, by hypertrophy, or expansion through replication of preexisting xcex2 cells. During embryogenesis, the xcex2-cell mass initially expands from differentiation of new cells, but by the late fetal stages the differentiated xcex2 cells replicate. Replication, then, is likely to be the principal means of expansion after birth, but the capacity to replicate appears to diminish with age. Adult islet cells have been shown to replicate by responding to stimuli known to initiate neonatal islet cell growth, e.g., glucose, growth hormone, and several peptide growth factors (Swenne, 1992; Hellerstrom et al., 1988; Bonner-Weir et al., 1989, Marynissen et al., 1983; Neilsen et al., 1992; Brelje et al., 1993). These observations suggest that the low level of xcex2-cell growth in the adult can accommodate functional demands. For example, during pregnancy or chronic obesity, xcex2 cell mass increases significantly yet is reversible since, following termination of pregnancy or after weight loss, an increased xcex2 cell death via apoptosis quickly reduces xcex2 cell mass.
It is generally accepted that all pancreatic endocrine cell types differentiate from the same ductal epithelium (Pictet et al., 1972; Hellerstrom, 1984; Weir et al., 1990; Teitelman et al., 1993), but whether they are derived from a common stem/precursor cell is uncertain. In normal adult pancreas, approximately 0.01% of the cells within the ductal epithelium will express islet cell hormones and can be stimulated to undergo morphogenic changes to form new islets, reminiscent of neogenesis. This neogenesis has been induced experimentally by dietary treatment with soybean trypsin inhibitors (Weaver et al., 1985), high levels of interferon-xcex3 (Gu et al., 1993), partial pancreatectomy (Bonner-Weir et al., 1993), wrapping of the head of the pancreas in cellophane (Rosenberg et al., 1992), specific growth factors (Otonkoski et al., 1994) and the onset of clinical IDD. Recently, attention has focused on the Reg gene (Watanabe et al., 1994, Otonkoski et al., 1994), identified in a subtracted cDNA library of regenerating rat islets, as a controlling element in the neogenesis of islet xcex2 cells. Up-regulation of the Reg gene (e.g., by hepatocyte growth factor/scatter factor) induces xcex2 cell proliferation resulting in increased mass, while down-regulation of the Reg gene (e.g., by nicotinamide) induces differentiation of the xe2x80x98pre-xcex2xe2x80x99 cells to mature cells. Thus, a population of precursor/stem cells remain in the adult pancreatic ducts and differentiation of this population can be evoked in vivo in response to specific stimuli. This action may actually occur continuously at low levels.
Although intensive efforts have been made to reproduce islet neogenesis in vitro, minimal success has been achieved. We now describe, for the first time, conditions which permit the growth and expansion of mammalian-derived islet-producing stem cells (IPSCs) in culture, as well as their differentiation to islet-like structures.
Numerous strategies (e.g., bone marrow replacement, immunosuppressive drugs and autoantigen immunizations) have been investigated as possible means to arrest the immunological attack against the pancreatic xcex2 cells. However, for these approaches to be effective, individuals who will eventually develop clinical disease must be identified. Most often, patients are identified too late for effective intervention therapy since the immunological attack has progressed to a point where a large percentage of the xcex2 cells have already been destroyed. Because the xcex2 cell is thought to be an end-stage differentiated cell, it was previously believed that the body has little capacity to regenerate new xcex2 cells, thus necessitating regular life-long insulin therapy. Recently, one approach to overcome this problem has been islet cell transplantation. Islet cell transplantation has the disadvantage that the islets are allogeneic which, in turn, can invoke an allo-immune response. Thus, there would be major advantages to growing islets of Langerhans containing functional xcex2 cells directly from IDD patients.
Recent observations of the Diabetes Control and Complications Trial (DCCT) that a tight control of glycemia can prevent or significantly reduce the incidence of the long-term complications associated with IDD (The Diabetes Control and Complications Trial Research Group, 1993) have shed light on the importance of the maintenance of the near-normal glucose levels in the periphery and the therapies that will lead to such a strict glycemic control. While intensive insulin therapy that achieves a tight glycemic control demands drastic changes in patient""s lifestyle along with increased incidence of hypoglycemic episodes, whole pancreas transplantation is known to render not only a tight glycemic control but also to substantially reduce secondary complications (Fioretto et al., 1998). However, the availability of both allogeneic pancreatic grafts and isolated islets is severely limited by donor availability (Teitelman et al., 1993). Recently, xenogeneic porcine islets have become a promising source of functional xcex2 cells, but require encapsulation to avoid autoimmune and xenoreactivities. The encapsulation by itself has not consistently provided protection of xenografts against autoimmune attack in nonobese diabetic (NOD) mouse model (Weber et al., 1997). Further, xenografts pose more serious issue of xenosis (introduction of animal pathogens into humans) (Bach et al., 1998). Thus, there is an urgent need for the development of methodologies to create a reliable and safer source (human) of islets, preferably generated in vitro in large numbers to the meet the demand for transplantation.
The subject invention concerns the discovery that islet-like structures containing insulin-producing xcex2 cells, as well as other islet cell types, can be grown in long-term cultures from pluripotent stem cells, i.e., islet producing stem cells or IPSCs. It also has been discovered that IPSCs may give rise to islet progenitor cells, IPCs. IPCs are pluripotent and committed to give rise to islet-like structures containing differentiated xcex1, xcex2, xcex4 and PP cells also found in in vivo islets of Langerhans. Islet-like structures are also referred to herein as IPC-derived islets (IdIs). IdIs contain xcex1 (or PP cells), xcex2 cells, and optionally xcex4 cells, each of which may be immature, and undifferentiated, proliferating cells.
The novel methods of the subject invention take advantage of the discovery that IPSCs exist even in the pancreas of adult individuals. To obtain IPSCs in vitro, a suspension of pancreatic cells can be cultured in a minimal, high amino acid nutrient medium that is supplemented with normal serum which is preferably derived from the same mammalian species which serves as the origin of the pancreatic cells (homologous serum). Several discrete phases of cell growth result in selection of IPSCs and subsequent progeny which are then induced to differentiate and form IPCs and ultimately IdIs, which are distinguishable from pseudo-islet or pseudo-pancreatic tissue of the prior art. In a first phase, a primary culture of pancreatic cells preferably including ductal epithelium is placed in a low serum, low glucose, high amino-acid basal medium. This culture is then left undisturbed for several weeks to permit establishment of a monolayer of ductal epithelium and to allow the vast majority of differentiated cells to die. Once this ductal epithelium layer is established, cell differentiation can be initiated by re-feeding the cell culture with the high amino acid medium supplemented with homologous normal serum plus glucose. After an additional period of growth, IdIs containing cells which may be immature and/or which may produce insulin, glucagon, somatostatin, pancreatic polypeptide (PP) and/or other endocrine hormones can then be recovered using standard techniques.
It was not previously known or suspected that pancreatic-derived non-islet cells (ductal epithelium) could be used to grow new IdIs, including xcex2 cells, in culture. The fortuitous discovery of culture techniques for growing IdIs in vitro eliminates what had previously been a substantial and longstanding barrier to diabetes research. The novel methods and materials described herein enable a better understanding of the mechanisms of diabetes. Furthermore, the ability to produce IdIs from IPSCs in culture now makes certain therapies for diabetes possible for the first time. For example, in accordance with the subject invention, IdIs obtained by culturing pancreatic tissue-derived IPSCs can be implanted in a patient as a way to control or eliminate the patient""s need for insulin therapy because the IdIs are able to produce insulin in vivo. The pancreatic tissue can be obtained from the prediabetic or diabetic patient, or from a healthy donor. Thus, the subject invention also concerns the use of the in vitro grown Idis of the subject invention for implantation into a mammalian species for in vivo treatment of IDD.
The subject invention also greatly facilitates genetic engineering of IPSCs or IPCs to resist subsequent immunological destruction. For example, the cultured IPSCs or IPCs can be transformed to express a protein or peptide which will inhibit or prevent the destructive immune process. Other useful proteins or peptides may be expressed. In addition, expression of specific autoantigens, such as GAD, 64 kD islet cell surface antigens (see Payton et al., 1995), or any other markers identified on the differentiated pancreatic cells, can be eliminated by standard gene knock-out or selection procedures to produce differentiated pancreatic cells which are not or are less susceptible to auto-immune attack. Methods for producing such mutant or knock out cells are well known in the art and include, for example, homologous recombination methods disclosed in U.S. Pat. Nos. 5,286,632; 5,320,962; 5,342,761; and in WO 90/11354; WO 92/03917; WO 93/04169; WO 95/17911, all of which are herein incorporated by reference for this purpose. In addition, a universal donor cell is produced by preparing an IPSC or IPC modified so as not to express human leukocyte antigen (HLA) markers as the cell differentiates into an IdI (see especially WO 95/17911).
Thus, the ability to grow functioning IdIs in vitro from the pancreatic cells of an individual represents a major technical breakthrough and facilitates the use of new strategies for treating and studying IDD. The discovery that IPSCs exist in the adult pancreas circumvents (without excluding) the need to use fetal tissue as a source of cells.
The subject invention also concerns the xcex1, xcex2, xcex4 and PP islet cells produced in vitro according to the methods described herein. These cells are produced from a mammalian pancreatic cell suspension cultured in vitro that gives rise to IdIs which contain the xcex1, xcex2, xcex4 and PP cells which may be immature.
The subject invention further concerns the in vitro growth, propagation and differentiation of IPSCs to generate IPCs, which in turn give rise to the formation of all of the differentiated types of cells that make up normal islets of Langerhans. Moreover, the subject invention concerns the in vivo use of in vitro grown IPSCs, IPCs or IdIs to produce a pancreas-like structure or an ecto-pancreatic structure that exhibits functional, morphological and histological characteristics similar to those observed in the endocrine tissue of a normal pancreas. Thus, a functional ecto-pancreatic structure grown in vivo from implanted ductal epithelium, IPSCs, IPCs and/or IdIs can be used to treat, reverse or cure a wide variety of pancreatic diseases that are known to result in or from damage or destruction of the islets of Langerhans.